Method of examining the identity of strains of lactobacillus fermentum

ABSTRACT

The invention relates to a pharmaceutical and/or dietetic composition for increasing the impact of the immune defense of higher living beings, wherein bacteria of the species  Lactobacillus fermentum  from at least one of the strains K1-Lb1 or K1-Lb6 or K2-Lb4 or K6-Lb4 or K7-Lb1 or K8-Lb1 or K9-Lb6 are contained in order to control the adaptive and natural immune defense by means of T helper 1 and T helper 2 cells and/or bacteria of the species  Lactobacillus fermentum  from at least one of the strains K2-Lb6 or K11-Lb3 are contained in order to strengthen the native immune defense.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of and claims priority to U.S. patentapplication Ser. No. 13/390,210, filed Apr. 23, 2012, which is a Section371 National Stage application of International Application No.PCT/DE2010/000953 filed Aug. 11, 2010 and published as WO 2011/018080 A2on Feb. 17, 2011, in German, which claims priority to German patentapplication Serial No. 10 2009 037 089.7, filed Aug. 11, 2009, thecontents of which are hereby incorporated by reference in theirentirety.

The invention relates to a pharmaceutical and/or dietetic compositionfor strengthening the effect of the human immune defence.

The immune defence is an ever present and active biological defencesystem of higher living organisms, which prevents tissue damage bypathogens. It consists of various organs as well as cells and moleculesof various types that form a complex network. It eliminatesmicroorganisms and other foreign substances that have penetrated intothe body and is also capable of destroying the body's own cells thathave become defective. The immune defence has great importance for thephysical intactness of living organisms, since virtually all organismsare continually subject to the influences of the living environment, ofwhich some even pose an existential threat.

If harmful microorganisms penetrate into the body, this can lead tofunctional disturbances and illness. Typical pathogens are bacteria,viruses and fungi as well as single-cell parasites, for example protozoaor other plasmodia, or multicellular parasites, such as tapeworms.

Currently new genera of harmful bacteria and other unwelcomemicroorganism are increasingly growing up, which are resistant not onlyto one but often even to a plurality of medicines, which are still veryeffective against the genera known hitherto. A threatening example inJuly 2009 is the pathogen N1H1, also known as swine flu, the mutants ofwhich could lead to a pandemic.

There is therefore currently increased interest in a strengthening andincreasing the effectiveness of the body's own defence.

Active substances with an immune modulating effect are appropriate forthis. The immune modulating effect can be distinguished into a so-callednatural or innate immunity, which a newborn baby already has, and theadaptive or acquired immunity, which only forms in the course of lifedue to contact with various invaders. Adaptive immunity makes use of theT helper cells, which are an essential part of the learning capacity ofthe body's defence system. Of the group of T helper cells, the T helper1 and T helper 2 cells are particularly important.

In the case of illnesses, a known and efficient treatment is toinfluence the so-called Th1/Th2 response such that the respectiveillness is combatted much faster and more extensively. To this end,their effect is increased by the administration of stimulatingsubstances.

Thus, the patent application KR 1007 42900 discloses the Lactobacillusrhamnosus DCC 3201 as a bacterial strain, which is added to foods as aprobiotic supplement in order to counteract and prevent atopicaldermatitis. Laboratory tests suggest that this bacterial strain acts toalleviate and prevent allergic reactions by maintaining the balance of Thelper 1 and T helper 2 cells.

The place where this bacterial strain was found in the feces of Koreanchildren should actually not be a disadvantage with appropriatepreparation according to the prior art, however, from the start, it doesnot arouse exclusively positive expectations.

In any case, this bacterial strain is not expected to provide an effectif the aim is a different, targeted shifting of the T helper 1 to Thelper 2 ratio, which acts on other illnesses.

Against this background, it is the object of the invention to findactive substances from a group which have an immune-modulating effect inthat they strengthen the immune responses of the body and in the processeither reinforce the natural immunity or support the adaptive immunityby stimulating T helper cells as directly as possible to increasedactivity and in the process selectively and primarily direct the effecteither to T helper cells 1 or to T helper cells 2.

As a solution, the invention provides a pharmaceutical and/or dieteticcomposition in which, for increasing the impact of the immune defence bymeans of T helper 1 and T helper 2 cells, bacteria of the genusLactobacillus fermentum from at least one of the strains K1-Lb1(deposition number DSM 22837) or K1-Lb6 or K2-Lb4 or K6-Lb4 or K7-Lb1(deposition number DSM 22831) or K8-Lb1 (deposition number DSM 22832) orK9-Lb3 and/or from the genus Lactobacillus plantarum of the strainK4-Lb6 (deposition number DSM 22830) and/or, for strengthening thenative immune response, bacteria from the genus Lactobacillus fermentumof at least one of the strains K2-Lb6 (deposition number DSM 22829) orK11-Lb3 (deposition number DSM 22838) are contained.

The designations KxLby used here are taken from the system used in theoriginal investigation of Kimere. K stands for Kimere, the source of thehitherto unknown strains presented here, Lb stands for Lactobacillus.From the large number of investigated species, some were chosen so thatthe first parameter of the designation—Kx—and the second parameter ofthe designation—Lby—do not contain sequential numbers.

The designations Kx-Lby of the strains of the genus Lactobacillusfermentum lead from two alternative kinds to an unambiguousidentification of the respective bacterial strain.

The first alternative is the known method of depositing a derivative ofthe respective genus at the DSMZ. Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH in Inhoffenstra[beta]e 7B, D-38124 Braunschweig.According to the procedure prescribed for a patent application, intactand living examples of the bacterial strains mentioned here weredeposited at the DSMZ, specifically on Aug. 6, 2009, under the followingfile numbers:

Lactobacillus fermentum K1-Lb1 (deposition number DSM 22837)

Lactobacillus fermentum K2-Lb6 (deposition number DSM 22829)

Lactobacillus fermentum K7-Lb1 (deposition number DSM 22831)

Lactobacillus fermentum K8-Lb1 (deposition number DSM 22832)

Lactobacillus fermentum K11-Lb3 (deposition number DSM 22838)

and Lactobacillus plantarum K4-Lb6 (deposition number DSM 22830)

The other strains have not yet been deposited at the DSMZ. For theirdisclosure, there serves the second alternative method of making knownthe PFGE band pattern.

An alternative for depositing living bacterial is clear identificationby means of pulse field gel electrophoresis pattern (PFGE). In theaccompanying FIG. 3, for each of the bacterial strains mentioned here,the so-called band patterns in each case, which are analysed by the PFGEmethod and therein show an identical band pattern, are therebyunambiguously identified as this strain of the respective genus.

The method of typifying microorganisms by PFGE has been known since 1984and is part of the recognized prior art. The researchers Schwartz andCantor have observed that, on application of electrical pulses, whichperiodically changed their orientation at a particular angle in ratio tothe agarose gel, large intact DNA molecules were isolated as bandpattern. This method is based on U.S. Pat. No. 4,473,452 and comprisesfour key steps, namely

1. Preparing the samples by culturing the microorganisms in a nutrientbroth, embedding the cells in gels and obtaining immobilized,deproteinized, intact DNA molecules

2. Specifying the progress of electrophoresis for an optimum separationbetween the molecules

3. Loading the samples into the gels and performing pulse-field gelelectrophoresis with the result of a typical band pattern

4. Analysing the band pattern by comparison with the band patterns ofsimilar genera of microorganisms. It is generally accepted in the priorart that the band pattern thereby obtained permits a clearidentification of the respective genus, somewhat comparable with afingerprint of a person.

The PFGE process, however, is not the actual content of this patentapplication but only one of two methods used for unambiguousidentification of the bacterial strains of the genus Lactobacillusfermentum to be protected. The common inventive feature of all genera isthe stimulating effect on the T helper cells.

The T helper cells belong to the lymphocytes, a sub-group of the “whiteblood corpuscles”, which are the most important agents of the humanimmune defence. An adult human has somewhat over a thousand differentlymphatic cells, which comprise about two percent of his bodyweight. Thelymphocytes, like all blood cells, originate from the bone marrow and,before their maturity into functioning cells of the immune defence, mustpass through additional development and differentiation stages.

Some of these precursor cells migrate from the blood-forming tissuedirectly into the thymus gland and develop into so-called T-lymphocytes.There, they learn, inter alia, to differentiate between the body's owncells and “invaders”. The remaining lymphocytes mature in the bonemarrow into so-called B cells.

The most important property of the lymphocytes is their capability ofreacting with a particular molecular shape according to the key-and-lockprinciple. To this end, as “lock” they bear on their surface receptors,which are specially adapted to the protein structure of one of millionsof different foreign bodies—the “keys”. Via the structure of the cell'sown protein and protein that is foreign to the cell on the surface ofthe macrophage, they recognize a “new invader” and mount the specificimmune defence.

For the actual execution of the immune defence, T killer cells arecreated, which selectively bind the invader and destroy it. Thestrengthening of this human immune defence by active substancesaccording to the invention takes place indirectly, namely viastimulating the increased formation of T helper cells, the Blymphocytes, to transform themselves into plasma cells, the body'santibody factories. In the course of their short lifetime of only a fewdays, they can pour out thousands of specialized defence molecules persecond.

It is at this point that the effect of the bacterial strains presentedhere starts, in that they act on one of the two types of T helper cells,either increasing the number of T helper 1 or T helper 2 cells, as aresult of which the number of the respective other kind is inevitablyreduced, and additionally or alternatively thereto, increasing thenumber of T helper cells that are grown overall.

By means of an additionally growing proportion of a very particular typewithin the group of T helper cells, their number with respect to thenormal state increases strongly, firstly due to the absolute increaseand secondly due to the increase of the relative proportion of the totalnumber of all T helper cells. As a result, a very much larger number ofplasma cells for defence molecules is very rapidly formed. Thus, thenumber of these defence molecules with a total of three factors isincreased, namely the absolute and the relative increase of the numberof T helper cells and their effect strengthening by multiplication inthe plasma cells.

A further, more advantageous strengthening factor for this effect isthat, due to the selection of one of the genera presented here, thoseplasma cells that form precisely the required antibodies arepredominantly formed. Due to the larger number of the bacterial strainspresented here, it is possible to select that bacterial strain thatforms antibodies that are particular effective for a particular type ofinvader. If, for example, a patient suffers from an allergy, it isappropriate to select one of the genera K1-Lb1 (deposition number DSM22837), K2Lb4, K4-Lb6 (deposition number DSM 22830), K6-Lb4 or K9-Lb3.

In addition, the immune system is prepared for a further contact withthe invader in that, the characteristic features of the invading foreignsubstance is stored in a portion of the B cells, so that, with the nextinvasion, the production of the suitable antibodies can start directly.This “secondary immune response” is the basis of most protectiveinoculations.

A further advantage of the large number of the bacterial kinds presentedhere is their multiple effect in multimorbid patients, that is to saypatients that suffer from more than one single illness. If, for example,the above-mentioned allergy patient additionally suffers fromhyperactivity of the gall bladder, the bacterial type K4-Lb6 (depositionnumber DSM 22830) is therefore of particular advantage, since it has aparticularly high bile salt resistance and, despite this second problem,therefore still has the first desirable antiallergenic effect.

Another advantageous property of the bacterial strains presented here istheir probiotic property. This property is proven inter alia by the factthat all strains were isolated from a food that has successfully beenused for generations, namely from Kimere, a dough produced from pearlmillet (Pennisetum claucum) by spontaneous fermentation, whose place oforigin is the district of Mbeere in Kenya. Since all types of bacterialare contained in a greater or lesser concentration in this widespreadand preferentially used staple food, their compatibility is proven. Itis also proven thereby that, in comparison to a very large number ofallopathic medicines, it can be classified as almost free of sideeffects.

The selection of bacilli of the genus Lactobacillus fermentumn wasinduced by the investigation of the typical conditions for thetraditional production of Kimere, namely the hygienic conditions duringthe preparation of the starting material pearl millet, its processing bywater treatment in very few vessels, which by our standards have onlybeen inadequately cleaned, its processing by grinding in a moist stateusually with one and the same mill, and its fermentation generally inone and the same vessel reserved for this purpose. That would, byEuropean standards, raise fears of contamination by microorganismsdisadvantageous to humans.

However, the investigation has shown that this is very clearly not thecase, but at least 90% of the microorganisms found belonged to the genusLactobacillus fermentum. From this it can not only be concluded thatthis genus is generally compatible with humans, but that it supresses,or for the most part eliminates, other microorganisms that are lesscompatible with humans.

In the studies, the population with microorganisms in the case of Kimerehas generally proved very stable, which is a positive feature for theuse of the kinds of bacteria found there. A further advantage is therelatively acid milieu, that is to say a relatively low pH. Since themicroorganisms have proven long-term stable in this acid milieu, theywill also survive the acid environment in the stomach and gut and inthis manner will prove effective on oral administration—for example asfood supplement.

Strains were even found that are characterised by such a high degree ofacid resistance that they pass through the stomach and small intestineeven into the large intestine in order to develop their healing effectthere.

Of the bacterial strains recognised as effective, almost all act notonly against a single type of complaint, but against a pluralitythereof, so that they are also suitable inter alia for treatment ofthose with multiple illnesses. However, clear focuses of the type ofeffect were found, so that the individual bacterial types can beselectively assigned to a focal action.

The first of the discovered types of effect is a strengthening of theimmune defence by a clear shifting of the T helper cells away from Thelper 2 cells towards T helper 1 cells. The strains K1-Lb1 (depositionnumber DSM 22837), K1-Lb6, K2-Lb4, K4-Lb6 (deposition number DSM 22830),K6-Lb4 and K9-Lb3 act predominantly with this focus. FIG. 1 shows theresult of the in vitro simulated effect. The different intensities ofthis type of effect can be read off in three stages.

These bacterial strains are thus suitable for medicines, foodsupplements or pharmaceutical preparations for the prophylactictreatment or reduction of the risk of the manifestation or for therapyof T helper 2-driven illnesses, such as, for example, eczema and/oratopical dermatitis and/or asthma and/or rhinitis allergica or otherallergies and/or tuberculosis and/or colitis ulcers and/or eosinophilicpneumonia.

These bacterial strains are thus suitable for medicines, foodsupplements or pharmaceutical preparations for the prophylactictreatment or for therapy in complaints, infections or other illnesseswhose defence is Th1 mediated, for example gut infections and/or traveldiarrhoea and/or colds and/or urogenital infections and/orHIV-associated complications and complaints and/or candidiasis.

Of the bacterial strains found, some act with a contrary accent of the Thelper cells: they effect a displacement of the response away from the Thelper 1 cells towards the T helper 2 cells. Thereby, bacterial strainsare presented which are also usable for entirely different illnesses inthe same advantageous manner. This effect predominantly characterisesthe strains K6-Lb2, K7-Lb (deposition number DSM 22831) and K8-Lb1(deposition number DSM 22832).

These strains which predominantly effect a reinforcement of the immunedefence by shifting away from the T helper 1 and towards the T helper 2responses, are suitable for producing a medicine or a food supplement ora pharmaceutical preparation for prophylactic treatment or for reducingthe risk of manifestation of autoimmune illnesses such as Crohn'sdisease or other T Helper 1-driven illnesses.

The aforementioned strains K6-Lb2, K7-Lb1 (deposition number DSM 22831)and K8-Lb11 (deposition number DSM 22832) also have an inhibiting effectagainst inflammation. And the K6-Lb4 strain also provesanti-inflammatory. These bacterial strains are therefore suitable forproducing a medicine or a food supplement or a pharmaceuticalpreparation for the reduction or elimination of the effects ofautoimmune illnesses such as arthritis or dermatitis or allergies orillnesses with an inflammatory component, such as a metabolic syndromeor arteriosclerosis.

The aforementioned strains K6-Lb2, K7-Lb1 (deposition number DSM 22831),K8-Lb1 (deposition number DSM 22832) and K6-Lb4 can be suitably andefficiently used for the prophylaxis or therapy of rheumatoid arthritis,hashimoto thyreoditis, uveitis, psoriasis, type 1 diabetes, sjögrendisease, coeliac disease, systemic lupus erythematosus, ankylosingspondylitis, Crohn's disease, inflammatory intestinal diseases,sclerodera, sarcoidosis, multiple sclerosis, vitiligo, Grave'sautoimmune thyroiditis, endocrinous opthalmopathy, myasthenia gravis,osteoarthritis, arteriosclerosis and therefore also cardiac infarction,other peripheral arterial occlusive diseases, cerebral infarction(stroke), metabolic syndrome and therefore also in the case ofadipositas, hypertonia, insulin resistance, type 2 diabetes,dyslipoproteinaemia, and amyotrophic lateral sclerosis, interstitialcystitis and/or irritable bowel syndrome (IBS).

Some of the bacterial strains presented here additionally strengthen theimmune defence in that they effect an increased release of defensin fromthe intestinal cells. It is also known that defensins carry manycationic and hydrophobic amino acid residues. They are thus amphipathicpeptides. These positive charges interact with the negative charges ofthe exciter membranes. The preference of the defensins is for membranesthat are characterised by a low proportion of cholesterol and therebydiffer from those of eukaryotic organisms. When they have penetrated themembrane, they also interact with anionic molecules within the pathogencell, such as DNA and RNA. By this means the action spectrum is broadand corresponds to that of a broad-spectrum antibiotic, so that it isdifficult for a pathogen to counteract the mechanism of the defensins.

To this extent, the effect mechanism is similar to that of the T helpercells, and they are therefore to be assigned to the same inventive idea.This type of effect applies predominantly to the strains K1-Lb7, K2-Lb6(deposition number DSM 22829) and K11-Lb3 (deposition number DSM 22838).

A further outstanding property is tolerance to a 3% bile salt solution.This feature applies particularly to the K4-Lb6 (deposition number DSM22830) strain. It ensures its survival through the greatest portion ofthe human digestive system as far as the large intestine. Because thebacterium is still in the living state when it arrives there it canstill develop its healing effect, namely the strengthening of the Thelper 1 reaction and the weakening of the T helper 2 influence.

For a very much larger number of the bacterial strains presented here,survival in the intestines is no longer possible, but survival in thestomach is. At least 8% of them survive a pH of 3.0 for at least 3hours. By this means they can still develop their effectiveness in fulleven after penetrating the pylorus within the stomach. This applies tothe strains K1-Lb1 (deposition number DSM 22837), K1-Lb6, K2-Lb4, K4-Lb6(deposition number DSM 22830), K6-Lb2, K6-Lb4, K7-Lb1 (deposition numberDSM 22831), K8-Lb1 (deposition number DSM 22832) and K9-Lb3.

These strains are therefore, inter aka, appropriate for prophylaxis forthe following complaints and illnesses, to avoid passing to a chronicstate and for system-related therapy of osteoporosis by acidification ofthe milieu and displacement of other microorganisms with the result ofimproved mineral and trace element resorption. Likewise, they areappropriate for liver failures and hepatic encephalopathy by reductionor inhibition of the resorption of toxins.

By means of these strains, lesser complaints with obstipation, irritablebowel syndrome and halitosis can be reached.

By means of the strains, the effects of lactose intolerance can besuccessfully suppressed by supporting lactose utilization and lactosedigestion.

Thanks to these bacterial strains, a positive influencing of the bileacid and cholesterol metabolism can also be achieved.

In a medicine or another form of administration, they are also helpfulagainst vaginosis or vaginitis, specific with oral or topicalapplication.

Another type of effect is the influencing of the concentration andcomposition at the membrane or wall of a bacterium, and consequently formodulation of the immune response of the recipient, at least one of thestrains expresses a glycosyl transferase or a sugar transferase or anacyl transferase or a lipoteichoic acid-exporting protein.

It is also observed as an effect mechanism that in the case of at leastone of the strains for glycosyl transferase, the sugar residue dockingcan be catalysed by the diacylglycerol synthesis (DAG).

Another subvariant is that for at least one of the strains, acyltransferases can be catalysed by the diacylglycerol synthesis (DAG).

In another variant of the effect of the bacterial strains presentedhere, for at least one of the strains, enzymes catalyse further sugartransfer for the synthesis of microbial peptidoglycans.

For at least one of the strains, the expulsion from the cytoplasm can bemediated by means of a lipoteichonic acid synthesis and/or by means of apeptidoglycan synthesis as transporter.

At the beginning of the description, it was mentioned that the bacterialstrains presented here are identified by the deposition of at least oneliving example with the DSMZ. Alternatively the respective band patternof the pulse field gel electrophoresis serves as the feature to beclearly identified. For this process, it is an appropriate intermediatestep to examine whether the effect-mediating genes have astrain-specific pattern of the suppressive subtractive hybridization(SSIt).

Methods and Special Features of the Underlying Study

1. Derivation from Kimere

Kimere is a spontaneously fermented dough or porridge from pearl millet,which is produced and consumed, inter alia in the district of Mbeere inKenya. East Africa, by a traditional method by first dry, then wet,milling on a millstone and subsequent fermentation.

A special feature is that Kimere is fermented 18-24 hours beforeconsumption and is assimilated in its actively fermented state as foodor is kept for up to three days. Otherwise it is similar to thepreparation of the normal East African porridge, called Uji. Kimere onlydiffers from Uji in its viscosity and consistency, since Kirnere is moreviscous.

The differences result from the manufacturing method, which influencesthe microbial population of the final product. The production of Ujiincludes the mechanical milling of maize, sorgum, millet or other cerealwith a hammer mill, followed by mixing the resulting meal with water andspontaneous fermentation or pouring back some of the already readyprepared Uji portion. Finally, the fermented product is boiled andsweetened before consumption.

In contrast thereto, Kimere is milled on a millstone in a dry state,followed usually by three wet milling operations with decanting andfinally a fermentation. In a variant, a portion of the product isconverted to a thin slime, to which water is added and then boiled. Theboiled Kimere is then mixed with the unboiled again.

The biggest difference from Uji is that Kimere is consumed in itsactively fermenting stage, and therefore contains living microorganisms,by contrast Uji is consumed immediately after boiling and does nottherefore contain living fermenting microorganisms. This method ofmanufacturing Kimere is typical of the regions of Mbeere, Tharaka, Chukand Embu east of Mount Kenya.

After the first purification process and the dry grinding, the brokengrain easily absorbs water—about 30% by volume—and is soaked for about30 to 60 minutes at room temperature. As a result, the broken grains aresoftened and prepared for the following wet grinding processes.

This is followed by a total of three wet grinding processes with a thickporridge as result. Between each wet grinding process, further water isadded and after each milling operation, decanting is performed. Theresult is a milky suspension, of which two thirds is boiled withstirring.

After boiling it is charged together with the further unboiled thirdinto a fermentation vessel and left to ferment for 18 to 24 hours. Theunbolted portion contains the microbes that were contained in the grainsand which have been absorbed during the milling operations.

Usually a portion of a previous preparation of Kimere is added, whichspeeds up the fermentation and influences the ultimate microbialpopulation. During fermentation, as a result of the lactobacilli, theproportion of phythates is reduced; pathogens are reduced and theflavour is improved.

The nutritional value of Kimere was thus improved compared tonon-fermented products and is free of coli bacteria and otherenterobacteria, which is an indication of microbial safety. The presenceof living bacteria in Kimere is of particular interest, since it is thebasis of a probiotic application.

2. Intake and Preparation of the Kimere Samples

The samples were taken in 11 yards in Kathera, a part of Kiang'Ombe, theEvurore part of the district of Mbeere. The samples were prepared andplaced overnight in fermentation vessels. On the following morning, theywere collected and taken to the Max-Rubner Institute in Kiel in a screwtop glass jar, where they were stored for a maximum of 24 hours at4[deg.] Celsius.

Transportation took less than eight hours, during which the samples wereconveyed at normal room temperature.

By this means, it was ensured that the isolated microorganisms behavedin a similar way to the microbial population during the otherwiseconventional fermentation, since Kimere is fermented for 18 to 24 hoursbefore consumption and can be consumed from the same fermentation vesselfor another three days thereafter.

The samples were very well mixed in a vortex mixer. 1 g of the samplewas added to 9 ml of sterile Ringer's solution to obtain a dilution of10<−1>. Then series of dilutions were made down to 10<−8> to count thelactobacilli.

3. Determining the pH of Kimere

To determine the pHi of Kimere samples, the method of the AOAC(Association of Analytical Communities) in the version of 1995 wasapplied. 10 g of the samples in each case was mixed with 40 ml ofdouble-distilled water; the mixture was measured after a waiting time of10 minutes using a Delta 320 pH measuring instrument. The pH values weremeasured in triplicate and the average value was calculated.

The aerobic mnesophiles were counted on a plate count agar (PCA) andaerobically incubated at 30[deg.]C. for 48 hours. The number oflactobacilli was determined by the area counting method on an MRS agar(Merck, Darmstadt, Germany) according to the process of De Man et. al.of 1960, and on the M17 agar (Merck, Darmstadt, Germany).

The MRS agar plates were incubated in an anaerobic chamber (MACSMG500+TG Airlock, dw-scientific, Shipley, West Yorkshire, England) at37[deg.]C. for 48 hours. The gas atmosphere consisted of 10% hydrogen,10% carbon dioxide and 80% nitrogen.

Then the M17 agar plates were aerobically incubated at 30[deg.]C. for 48hours. Only plates with colony-forming units (cfu) between 10 and 300per grain were analysed and the result was plotted as a decadiclogarithm of the cfu number per gram of the wet weight of the sample.

4. Isolation and Biochemical Characterization of the Lactobacilli

1 g of the sample of Kimere was weighed in 9 ml Ringer's solution and aplurality of tenfold dilutions were prepared down to 10<−8>. Of eachdilution of 10<−5> to 10<−8>, 0.1 ml in each case was spread two fold onan MRS agar plate. The plates were incubated at 37[deg.]C. for 48 hoursin an aerobic chamber as described above.

Individual, different colonies were chosen based on their morphology andpurified by spreading again on MRS agar, and inspected with themicroscope. The gram staining and the catalase reaction were performedas described by Harrigan and McCance 1990.

All rod-shaped gram-positive and catalase-negative isolates were storedat −80[deg.]C. in the Cryo-Bank® vials from Germany for furthercharacterizations. The production of CO2 glucose in MRS solution wasdetermined by means of Durham tubes. The chemical characterisation ofthe chains was performed with API 50 CH kits with API 50 CHL medium inconformity with the guidelines of the manufacturer Biomrieux,Niirtingen, Germany, and by the fermentation of various carbohydrates inMRS solution. The isolates were also tested for their capability forgrowth at 15[deg.]C. and 45[deg.]C.

5. Molecular Characterisation of the Lactobacilli by Means of theAmplified Ribosomal DNA Restriction Analysis (ARDRA)

The DNA material for the polymerase chain reaction (PCR) was preparedaccording to the method of Ismail 2007 and Vaneechoutte et. al. 1992: Acolony from an MRS agar plate was dissolved in 500 [mu]l of PCR buffersolution to produce a turbid suspension (McFarland 3) and incubated in aThermomixer at 95[deg.]C. for ten minutes. This DNA material was storedat −20[deg.]C. until the PCA analysis was performed.

The PCR was performed in an Eppendorf Mastercycler 5330, Hamburg,Germany, in volume units of 50 [mu]l, consisting of 20 [mu]l PCR mastermixture (Fermentas, Sankt Leon-Roth, Germany), 26 [mu]l double-distilledwater, 1 [mu]l of the respective primer and 2 [mu]l of the DNA sample.The PCR program was Initial denaturisation at 93[deg.]C. for fiveminutes, 35 cycles of denaturisation at 92[deg.]C. for one minute,hardening at 58[deg.]C. for 1.5 minutes and elongation at 72[deg.]C. for2.5 minutes respectively, followed by a final elongation at 72[deg.]C.for ten minutes and cooling and keeping at 4[deg.]C.

These sequences of oligonucleotide primers were: forward-UP68 5′-TGG CTCAGA TTG AAC GCT GGC GGC-3′ and reverse-UP69 5′-CCT TTC CCT CAC GGT ACTGGT-3′. An amplification of about 2.4 kb was produced, which for themost part consisted of the 16S rDNA, 16S-23SrDNA spacer region andportions of the 23SrDNA-stand (Ismail, 2007, Vaneechoutte et al 1992).The restriction was performed overnight at suitable temperatures in 20[mu]l volume units, which contained 0.5 [mu]l restriction enzyme(HaelII., Hinfl.) (Fermentas) DdeI (New England Bio Labs Inc.), 2 [mu]lenzyme buffer solution (Buffers R for HaellI. and Hinfl and Buffer 3 forDdeI), 2.5 [mu]l-15.5 MI 1*TE buffer, and 2.5-15 [mu]l PCR product.

The used volume was based on the strength of the signal from the PCRproducts, which were analysed on the pattern of the agarose gelrestriction fragment by electrophoresis in 1.5% agarose gel in 1*TAEbuffer at 80 V for two hours. Staining was performed with ethidiumbromide for 30 minutes, followed by washing for 15 minutes in water. Thephotographs were taken under UV light.

6. Strain-Specific PCR for Lactobacillus fermentum

Strain-specific PCR was performed according to the method described byDickson et. al. 2005. Primary LF1 (nt196-215; 5′-AAT ACC GCA TTA CAA CTTTG-3′) and LF2 (nt529-510; 5′-GGT TAA ATA CCG TCA ACG TA-3′) were usedspecifically for Lactobacillus fermentum. An amplification of 337 bplength was produced. The amplification program was used without change.The PCR products were analysed as described for ARDRA.

7. 16S rDNA Sequences:

Based on the ARDRA profile data in each case (chart 2-2), 12 strainswere chosen for sequencing. The strains were chosen based on theprofiles that had been chosen after application of the Hinfl restrictionenzyme. Five strains that represented each of the two ARDRA profiles (A1and A2) and one of the profiles B and C in each case. The PCR productsgenerated with the primers UP68 (forwards) and UP 69 (backwards)(Vaneechoutte et. al. 1992), were purified with a clean up kit(NucleoSpin® Extract II, Macherey-Nagel, Dtiren, Germany) according tothe manufacturer's operating instructions. Purification was controlledby electrophoresis in 1% agarose gel. Partial sequencing of 16S rDNA wasperformed using UP68 as sequencing primer. Sequencing was performed atMWG Biotech AG in Ebersberg, Germany.

8. Pulse Field Gel Electrophoresis (PFGE)

PFGE analysis of lactobacillus fermentum strains was performed accordingto the method of H-oppe-Seyler et al. 2003. The restriction enzymes thatwere tested were NotI, AscI and Sinai. AscI gave the best profiles andwas used in this study. Electrophoresis was performed in a Bio-RadCHEF-DRII system which was adjusted to the following conditions:Temperature 14[deg.]C., pump rate 70 rpm, first switching time 2.0seconds, final switching time 30 seconds, running time 24 hours andvoltage 175 volt. After electrophoresis, the gel was soaked in ethidiumbromide (0.5 mg/l) for 30 minutes. The gel was then washed in distilledwater for 10 minutes and photographed under UV light.

9. Data Analysis

The microbiological quality of the colony-forming units (cfu) and the pHwere analyzed using SAS version 9.1. The ARDRA gels were visuallyanalysed and the patterns compared with those of typical strains andbrought to agreement. The orientation of the 16S rDNA sequences and thegeneration of the dendogram were based on Escherichia coli ATCC 1 775T(access number X80725) as a root of the dendrogram (Tamura et. al. 2007)with the MEGA4 neighbour-joining method.

The sequences were also compared with the sequences in the gene bank forthe DNA data using the BLAST algorithm (Altschul et. al. 1997). The PGGEprofiles were analysed using the GelCompar 11 program (Applied Maths,Kortrijk, Belgium). Similarities between different chains were derivedusing Dice's coefficient. The unweighted pair group method witharithmetic mean (UPGMA) was used to cluster different profiles.

10. Microbiological Quality of Kimere

Lactobacilli make up the biggest portion of the microbial population offermented Kimere. Coli bacteria and other enterobacteria were detectedneither in 0.1 gram of the sample in testing in LST nor in 0.01 gram ofthe sample on VRB agar, nor in 0.01 gram of the sample on VRBD.

11. Biochemical Characterization of the Lactobacilli

48 strains of isolated lactobacilli were examined for growth at growthat 15[deg.]C. and at 45[deg.]C. and for their gas production fromglucose and for the capability to metabolize various sugars. Inaddition, they were characterized by API 50CHL. All strains with theexception of K1-Lb5 were capable of growing on melibiose and raffinose.All strains with the exception of the isolated K4-Lb6 (deposition numberDSM 22830) were capable of growing on mannitol, melecitose, rhamnose,sorbitol and trehalose. Only three isolates were capable of growing oncellobiose. Most strains with the exception of K4-Lb6 (deposition numberDSM 22830) were capable of growing at 45[deg.]C.

By the identification according to API 50CHL, 33 strains ofLactobacillus fermentum could be isolated. The API identity pointsextended from 38.9 to 99.9%.

12. PFGE Profiles of the Kimere Isolates

All isolates from Kimere, which was identified as Lactobacillusfermentum, were subjected to PFGE analysis. Of the restriction enzymesthat were used for testing (AscI, NotI, SmaI), only AscI producedresults suitable for comparison. FIG. 3 shows the profiles of the 10strains disclosed here. Although they have only been isolated from asingle kind of fermented food, most kinds show fairly different PFGEpatterns, which illustrates the high biodiversification within thestrains of Lactobacillus fermentum from Kimere.

The very significant differences of the band patterns between theindividual strains also make it plausible that a clear identification ispossible using this band pattern.

12. Strain-Specific Immunomodulation of the Strains of LactobacillusFermentum Isolated from Kimere

It indicates that probiotic effects are strain-specific. So far, veryfew studies have compared strains of the same genus to reach thisconclusion. With the study described here, the probiotic potential ofstrains of the genus Lactobacillus fermentum are investigated anddemonstrated strain-specifically in vitro. Nine strains of Lactobacillusfermentum and one strain of Lactobacillus plantarum, which could grow ina 3% bile salt solution, were selected from 48 strains by a biletolerance test.

These strains were further investigated, namely for their growth rate inan MRS medium (Man Rogosa and Sharpe), supplemented with 0.3% and 3%bile salt solution and for their resistance to a low-pH by counting thesurviving colony-forming units (cfu) after exposure to pH 2 and pH 3 forthree hours.

The immune-modulating potential was investigated by co-incubation of thestrains with human peripheral blood mononuclear cells (PBMCs) for 48hours at 3TC and measurement of the cytokines, specifically for Th 1cytokines (interferon-[gamma]) and Th2 cytokines (Interleukin-4) in thesupernatants by enzymatic-linked immunosorbent assay (ELISA).

All strains could tolerate 0.3 bile acid, with strain K4-Lb6 (depositionnumber DSM 22830) even withstanding 3.0% bile acid. The K7-Lb1(deposition number DSM 22831) and K8-Lb1 (deposition number DSM 22832)strains even showed a survival rate of about 53% and 27.2% at a pH of2.0; by contrast most strains only remained intact at a pH of 3.0.

As with LGG, the strains K7-Lb1 (deposition number DSM 22831) and K8-Lb1(deposition number DSM 22832) reduced interleukin-4. LGG and all otherstrains induced a changed from the basal Th1/Th2 ratio towards Th1 withthe exception of the strains K7-Lb1 (deposition number DSM 22831) andK8-Lb1 (deposition number DSM 22832) induced a change towards Th2. TheSEA-stimulated Th1/Th2 ratio was reduced from the Lactobacillusfermentum strains, while, by contrast, it was raised by LGG.

The study thus shows strain-specific peculiarities of the strains ofLactobacillus fermentum and also shows the capability of suppressing theproduction of interferon-[gamma] induced by Staphylococcus entertoxin A(SEA), a property that has not been reported by earlier studies ofLactobacillus fermentum and of most other Lactobacilli. These variousproperties can be used to combat illnesses that are driven by a Th1response or a Th2 response.

13. Fermented Probiotic Food Supplements and their Effect on the ImmuneSystem

The application of lactic acid bacteria (LAB) for human nutrition hasbeen known for millennia-wherever fermented milk or other fermentedfoods have been enjoyed. Their direct association with human health wasfirst observed in 1908 by the Russian Nobel laureate Elie Metchnikoff,who ascribed the health and longevity of Bulgarian herdsmen to themicroorganisms in the yoghurt that they frequently consumed.

The observations found new interest when the use of Lactobacilli andBifidobacteria gained importance as food supplements. “Probiotic” isdefined as “A live microbial feed supplement which beneficially affectsthe host animal by improving its intestinal microbial balance.” (Filler,R. 1989, Probiotics in Man and Animals. Journal of Applied Bacteriology66, 365-378).

The definition and application of “probiotics” continued to develop assoon as scientific evidence had been obtained (Schrezenmeir, J. and deVrese, M., 2001, Probiotics, Prebiotics and Synbiotics, approaching adefinition. American Journal of Clinical Nutrition 73, 361-364.) A morerecent proposal by Galdiano, C. M., de LeBlanc, A. D., Vinderola, G.,Bonet, A. E. B, and Perdigon, G., 2007, Proposed Model: Mechanisms ofirnmunomodulation induced by probiotic bacteria. Clinical and VaccineImmunology 14, 485-492) includes immunomodulation in the definition ofprobiotics. They defined probiotic as “living microorganisms that, whenadded to foods, influence the composition and activity of the microbesin the digestive system, modulate the inflammatory reaction, improve thenon-specific barriers in the gut and strengthen or improve the immuneresponse of the mucosae and of the system.”

The selection of strains for use as probiotics concentrates on two keyproperties: The adaptability of the strain itself and thehealth-supporting or functional aspect. These selection schemes include:Survival in a milieu with low pH, growth in the presence of bile salts,adhesion to epithelial cells on the intestinal wall, colonization withinthe digestion system, maintaining the microbial equilibrium,non-pathogenicity towards the recipient, resistance to technical stressduring processing and distribution and, finally, the capability toimprove the health of the recipient. (FAO/WHO, 2002, Guidelines for theevaluation of Probiotics in food. Joint FAO/WHO Working group report onDrafting Guidelines for the evaluation of Probiotics in foodftp://ftp.fao.org/esesn/food/wgre-port2.pdf, 1-11).

It must be noted here that not every individual probiotic strain mustshow all these properties. According to recent scientific findings,bacteria must not be “living” to product immunomodulating effects sinceboth living and dead bacteria or only bacterial DNA demonstrably showssome health benefits.

However, survival in the digestion system is essential to ensure thatthe probiotics reach the target in the active state, depending on thefunctional requirements such as colonization of the digestion system andthe exclusion of putrefactive bacteria or pathenogens.

Immunomodulation by probiotics is achieved by interaction with theimmune cells of the recipient and the secretion of various signalmolecules, such as cytokines and immunoglobulins. The T helper cellsperform an outstanding role within the immunocompetent cells in theimmunomodulation, which produces cytokines by means of probiotics.

In interaction with the antigens, available T helper cells differ invarious ways, depending on the type and number of the antigens. Theenvironment of the cytokines ultimately determines the familyrelationship and the effect profile. The differentiation into T helper 1cells depends on the interleukin 12-effected activation of transcription4 (STAT4), which leads to interferon-[gamma] production, which, via aseries of cascades, leads to a further growth of T-bet-guidedtranscription factors, which leads to a further growth ofinterferon-[gamma], interleukin-12 receptors [beta]2, interleukin-4supression and the maintenance of family relationships to T helper 1cells.

On the other hand, T helper 2 cells differ in the presence ofinterleukin-4 by STATE, an increase of interleukin-4 production, thesuppression of interferon-[gamma] production and thus maintenance of Thelper 2 descent.

The other kind of differentiation of T helper cells is T regulation(Treg), which produces interleukin 10, Treg cytokines IL-10 regulate theimmune system both due to the suppression of T helper 1 cells as well asof T helper 2 cytokines by ensuring peripheral tolerances. The fourthknown kind of natural T helper cells is differentiation via interleukin23, which leads to the production of interleukin 17.

It is assumed that a disequilibrium between T helper 1, T helper 2, Tregand T17 cells is the precursor to a manifestation of variouspathological conditions. The probiotic function increases cytokineproduction towards a reinforcement of the immnune defence, a reducedallergic or autoimmune reaction and/or a lower inflammation reaction.

A modulation of cytokine production by probiotics in human and animalmodels was found both in vitro (Pochard, E., Gosset, P., Grangette, C,Andre, C, Tonnel, A. B., Pestel, J., Mercenier, A., 2002, Lactic acidbacteria inhibit T(H)2-cytokine-production by mononuclear cells fromallergic patients. Journal of Allergy and Clinical Immunology 110,617-623, and Ghadimi, D., Folster-Holst, R., de Vrese, M., Winkler, P.,Heller, K. J., Schrezenmeir, J., 2008, Effects of probiotic bacteria andtheir genomic DNA on THI1/TH2 cytokines production by peripheral bloodmononuclear cells (pbmcs) of healthy and allergic subjects,Immunobiology 213. 677-692) and in vivo (Kopp, M. V., Goldstein, M.,Dietschek, A., Sofke, J., Heinzmann, A. and Urbanek, R., 2008.Lactobacillus GG has in vitro effects on enhanced Interleukin-10 andInterferon-[gamma]-release of mononuclear cells but no in vivo effectsin supplemented mothers and their neonates). Clinical and ExperimentalAllergy 38, 602-610).

In most cases, a mixture of Th1, Treg and Th2 cytokines was detected,although several authors include an exchange of Th1 or Th2 in the actionsequence. This immunomodulation depends on the dose and time and alsoshows dependencies on the inoculation of the strains.

14. Bacterial Cultures

The bacterial strains used in this study are isolated from Kimere, afermented dough from Kenya and characterised according to molecularmethods and kept at −80[deg.] Celsius in a MAST Cryobank® (MASTDiagnostic, Reinfeld, Germany). As positive comparison controls for Thelper 1 cytokines and T helper 2 cytokines, the strains Lactobacillusrhamnosus GG (ATCC 5310, a strain that is known to stimulateinterferon-[gamma]) and Escherichia coli TG1 (BU-00035, a strain that isknown to stimulate interleukin-4). They were procured from commercialsources.

The Lactobacilli were cultured anaerobically overnight in MRS solutionat 3TC in an MAC8-VA500 workstation (Don Whitley Scientific Limited,UK). The Escherichia coli TG1 were cultured aerobically overnight at37[deg.] in a Luria-Bertani (LB) solution. The cells were centrifugedfor two minutes at 14,000 g (corresponding to 144,500 rpm on anEppendorf Minispin Plus centrifuge). The bacterial pellets were washedtwice with a phosphate-buffered salt solution (PBS) dissolved in 1 mlPBS, containing 20% glycerine, then counted in an improved Neubauer typehemocytometer and subsequently brought to a concentration of 10<8> cellsper millilitre in a PBS solution with 20% glycerine and kept at−80[deg.] Celsius until use.

15. Resistance to Bile Salts

48 isolates of Lactobacilli were tested for their resistance to bilesalts according to the above-described method with small changes. 0.1 mlof an MRS growth culture was added to 9.9 ml of MRS solution with 0.3 or0.5 or 1.0 or 2.0 or 3% cattle bile (Siegmar). They were incubated for24 hours at 37[deg.] Celsius. The growth was recorded after visualchecking of the turbidity of the tubes.

Ten isolates were selected, which even showed growth with 3% ox bile.Their growth was then monitored with 0.3% and 3% ox bile. 2 ml asaliquot (partial sample) of Lactobacilli from a fresh culture grownovernight was suspended in PBS as spheres and 100 [mu]l thereof wasinoculated into an MRS solution, to which 0.3% (w/v) bile solution hadbeen added. A 0.3% (w/v) bile salt concentration has been classified asphysiologically relevant and in many studies serves for probioticselection. The same applies to a 3% ox bile (w/w).

The cultures were incubated in the water bath at 37[deg.] Celsius. Thegrowth was observed hourly by measurement of the absorption at 620 nm (AIndex 620 nm). The tests were repeated 3 times. The absorption valueswere recorded over the incubation times as described by (Gilliand andWalker. (Gilliand, S. E. and Walker, D K 1990, Factors to consider whenselecting a culture of Lactobacillus acidophilus as a dietary adjunct toproduce a hypocholesterolemic effect in humans, Journal of Dairy Science73, 905-911).

16. Acid Resistance

2 ml of a culture grown overnight was centrifuged for one minute at14,000 g (corresponding to 145,400 rpm on an Eppendorf Minispin Pluscentrifuge). The pellets were washed twice with 2 ml Ringer's solutionand suspended again in 1.5 ml Ringer's solution. 0.1 ml of thesuspension was transferred to 5 ml of 0.35% NaCl solution (supplementedwith pepsin (0.1 gram per 50 ml) and adjusted to the various pH values2.0 and 3.0 and 6.5 with HCl), followed by incubation at 37[deg.]Celsius in a water bath.

After three hours, 5 ml of 0.1 M phosphate buffer solution with a pH of6.5 was added. The survival rates were detected by counting on MRS agar.The colony-forming units (cfu) were expressed as a percentage of thecontrol values that had been obtained on incubation with a pH of 6.5.

17. Test of Immunomodulation with PBMCs (Peripheral Blood MononuclearCells).

Complete blood was collected from healthy donors aged between 21 and 52years and mixed with EDTA (ethylenediaminetetraacetic acid) for purposesof anticoagulation. Individuals who had reported an allergy or hadrecently suffered infections of the respiratory passages or had takenmedication were excluded. The enlisting of test subjects and taking ofblood samples was performed according to strict ethical points of viewwhich had been laid down by the ethics committee of the University ofKiel for the use of human test subjects in research. A written agreementwas obtained from all test persons before their registration for thisstudy. The samples were kept at room temperature (18-25[deg.] Celsius)until isolation of the PBMCs.

The PBMCs were isolated as described above. Blood was mixed with sodiumchloride (NaCl) and filled into 50 ml centrifuge tubes at a mixing ratioof 1:1 and placed on a Ficoll Lymphoprep Plus (Axis Shield PoC AS, Oslo,Norway) at a ratio of 1:2 (16 ml Ficoll and 32 ml blood) aftercentrifulging at 179.5 g (1,300 rpm) on an Eppendorf centrifuge 5810 R,r=9.5 cm) for 30 minutes at 18[deg.]C. The PBMC layers were filled intosterile centrifuge tubes with 50 ml content. Washing liquor (PBS plus10% FCS) was added to fill the tubes (about 25 ml) and then tocentrifuge them at 179.5 g for 10 minutes at 4[deg.] Celsius.

Washing was performed three times with PBS plus 10% FCS; each time, halfof the washing medium was discarded of and finally the entire washingmedium was removed. After the third washing, the PBMCs were suspended inthe complete medium (RPMI 1640 Medium+10% FCS+1%(penicillin+streptomycin)) all supplied by Gibco, Karlsruhe, Germany.The PBMCs were counted in a hemocytometer, 10 [mu]l:90 [mu]l,PBMCs:staining agent and expressed as PBMCs/ml of the medium.

18. Coincubators of the PBMCs and Bacteria:

The concentration of PBMCs that was used was 1*10<8> lines/ml and thebacteria 2*10<7> cells/ml (1:20 PBMC to bacteria ratio). Besides theunderlying condition (medium), with the use of Staphylococcus entertoxinA (SEA), a stimulating state was measured at a concentration of 2[mu]g/ml. The treatments were: PBMC's+medium as comparison parameter;PBMC's+SEA+medium; PBMC's+bacteria+medium and PBMCs+SEA+bacteria+medium,in each case in 1 ml volume units in a plate with 24 wells and induplicate. After incubation at 37[deg.]C. for 48 hours, the supernatantwas centrifuged at 179.5 g, 4[deg.] Celsius for ten minutes and thenfiltered through a sterile microfilter with 0.2 [mu]m mesh size, andfilled into Eppendorf tubes (1.5 mi) in volume units of 250 [mu]l andkept at −20[deg.]C. for the ELISA test.

19. Cytokine Sample

Examination of the detection of interleukin and interferon-[gamma] inthe supernatants was performed with an ELISA plate with 96 wells,corresponding to the manufacturer's directions. All ELISA reagents werepurchased from Mabtech AG, Hamburg, Germany. The lower detection limitswere 1 pg/ml for interleukin and 4.2 pg/ml for interferon-[gamma], asrecommended for each kit.

The absorption was measured at 450 and 570 nm wavelength in a microplatereader, Molecular Devices, Munich, Germany. For correction of thewavelength, the reading of the optical density at 570 nm was subtractedfrom the value read for 450 nm. The mean absorption values of the emptyplate (background control) was deducted from the standard values for thestandard test specimens and the values of the samples, and then astandard curve was generated using the curve fitting program with fourparameters and the derivation of the cytokine concentration in thesamples.

20. Statistics

By means of ANOVA, the mean values of the bile resistance of variousstrains were compared. For the data for immunomodulation, the analysiswas performed in two steps: First, all the strains and the comparativetest samples were compared with one another. In the second step, thevalues of the control parameters LGG and TG1 were excluded, which areclassified as “blips” for interferon-[gamma] and interleukin-4.

With the general linear model of the SPSS 9.0 simulation software, themean values of the SEMs were derived and the mean values of differentstrains were compared using the “Mann-Whitney U test”. With Sigma Plot11.0, the results were presented graphically. Statistical significancewas considered at P>0.05. To define the direction of the displacement ofthe ratio of T helper 1 to T helper 2 values in the immune response, theratio of interferon-[gamma] to interleukin-4 was used. The mean ratiowas defined at 100% and the values for the other test specimens wereexpressed as a percentage of this comparison value. Values over 100%were classified as a T helper 1 shift and values smaller than 100% as aT helper 2 shift.

21. Resistance to Bile Salts and Low pH

To check their bile tolerance, all 48 isolates were administered to anMRS medium overnight, which was adjusted to 0.3%, 0.5%, 1%, 2% and thento a proportion of 3% ox bile (w/v). By checking the average turbidity,the growth of, first, 48, then 28, 26, 21 and 12 isolates wasascertained (see FIG. 4A).

The monitoring of their growth movement (change in OD620 nm) in MRSsolution, supplemented with 0.3% ox bile (w/v) shows a good growth ofall strains, which was indicated by a short delay phase (FIG. 4B). Onlythe strains K1-Lb6 and K8-Lb1 (deposition number DSM 22832) requiredover three hours to reach 0.3 units of A620 nm. As shown in FIG. 4C, thegrowth of the strain K4-Lb6 (deposition number DSM 22830) differedsignificantly from the mean value of the others in a solution with 3% oxbile (w/v). The strain K4-Lb6 (deposition number DSM 22830) began togrow almost without a delay phase. For all the other strains, the growthwas only visible after a prolonged incubation overnight.

If the strains were subject to a pH of 2 and a pH of 3 for three hours,they showed the different stages of survival as shown in FIG. 5. At a pHof 3, four strains, namely K1-Lb6, K7-Lb1 (deposition number DSM 22831),K8-Lb1 (deposition number DSM 22832) and K9-Lb3, showed survival ratesof over 80%. Two of these, namely the K7-Lb1 (deposition number DSM22831) and K8-Lb1 (deposition number DSM 22832) strains, showed thegreatest stress resistance towards most acids with survival rates of52.9% at pH 2.0 and 27.2% at pH of 6.5, compared to the control strains.

22. Cytokine Production Pattern

The Kimnere strains supressed the SEA-induced interferon-[gamma]production, a phenomenon that has so far not been observed for strainsof Lactobacillus fermentum. The Kimere strains suppressed interleukin-4production. In the case of the strains K7-Lb1 (deposition number DSM22831) and K8-Lb1 (deposition number DSM 22832), the interleukin-4 wasreduced to close to the limit of detectability (FIG. 6B).

A strain-dependent shift of the interferon-[ganunmma] to interleukin-4ratio, and therefore of the accordingly proportional ratio of T helper 1to T helper 2, was observed (FIG. 6C).

The strains K7-Lb1 (deposition number DSM 22831) and K8-Lb1 (depositionnumber DSM 22832) displaced the T helper 1 to T helper 2 balance towardsT helper 2, while the others induced a change towards T helper 1. Allthe tested strains were, in comparison to TG1-which is known to suppressinterleukin-4 production-capable of significantly suppressing theproduction of interleukin-4. Compared to the control substances, thestrains K6-Lb4 and K8-Lb1 (deposition number DSM 22832) suppressed thebasal secretion of interleukin-4 (FIG. 6B).

K7-Lb1 (deposition number DSM 22831) and K8-Lb1 (deposition number DSM22832) suppressed the SEA-induced interleukin secretion (FIG. 6B).Interleukin-4 extended from 1.96 pg/ml for K8-Lb (deposition number DSM22832) (almost not detectable) to 45 pg/ml for K1-Lb1 (deposition numberDSM 22837) (FIG. 6B).

The basal interferon-[gamma] production was significantly suppressed bythe strains K7-Lb1 (deposition number DSM 22831) and K8-Lb1 (depositionnumber DSM 22832) (FIG. 6A).

A suppression of SEA-induced interferon-[gamma] production by the testedbacterial strains could be observed. It was particularly pronounced forthe strains K7-Lb1 (deposition number DSM 22831) and K8-Lb1 (depositionnumber DSM 22832), but also for all other Kimere strains (FIGS. 6A to6C).

Since the overproduction of interferon-[gamma] plays a leading role inthe pathenogenesis of IBD, the reduction of interferon-[gamma] by thestrains of Lactobacillus fermentum from Kimere presented here shows thatthese strains have an immunomodulating property, which can be used inthe battle against illnesses that are dependent on T helper 1 cells.Various strains of Lactobacillus fermentum from Kimere induced either aT helper 1- or a T helper 2-driven response, depending on the respectiveimmunomodulation.

The various strains have different stimulation effects oninterferon-[gamma] and interleukin-4 (FIG. 6). The strains' ability tosuppress the basal interleukin-4 formation shows their possibleapplication for T helper 2-driven illnesses.

The ability of Lactobacillus fermentum isolates from Kimere to suppressthe formation of SEA-induced interferon-[gatmma] and simultaneously toblock the production of interleukin-4 shows unique possibility in thesuppression of autoimmune illnesses and atopic illnesses withinflammatory components.

Further details and features of the invention are explained below ingreater detail with reference to graphical descriptions. However, theyare not intended to limit the invention but only explain it. Inschematic, view:

FIG. 1 shows an overview of the properties of the individual strains

FIG. 2 phylogenetic tree of the Lactobacilli presented here incomparison with known bacteria

FIG. 3 band pattern of all 10 strains according to the PFGE process

FIGS. 4A-4C tolerance of the strains to bile salt

FIG. 4A: shows the number of the surviving strains on exposure todifferent concentrations of cattle bile.

FIG. 4B: shows the measurement of the absorption of different strains at620 nm in 0.3% concentration of cattle bile solution.

FIG. 4C: shows the measurement of the absorption of different strains at620 nm in 3.0% concentration of cattle bile solution.

FIG. 5 resistance of the strains to very acid milieu

FIGS. 6A-6C Th1 and Th2 cytokine production of human blood cells (PBMCs)after coincubation with the strains

FIG. 6A: shows the production of interferon-γ in different strains.

FIG. 6B: shows the production of interleukin-4 in different strains.

FIG. 6C: shows the resulting ratio of interferon-γ and interleukin-4.

FIG. 1 shows, as a key property of the strains presented here,characteristic effects and their intensities. It is clear that almostall strains, with the exception of K11-Lb3 (deposition number DSM22838), show three characteristic properties that distinguish them fromone another. All of these strains have a more or less high bile salttolerance and a greater or lesser pH tolerance. All the strains have animmunomodulating effect. This immunomodulating effect belongs either tothe natural or innate immunity, such as the elevated defensin releasedue to the strains, in this case K2-Lb6 (deposition number DSM 22829)and K11-Lb3 (deposition number DSM 22838), or is part of the adaptive oracquired immunity, such as the influencing of the Th1/Th2 response.

FIG. 1 shows that, of the strains presented here, K1-Lb1 (depositionnumber DSM 22837), K1-Lb6, K2-Lb4 and K4-Lb6 (deposition number DSM22830), strengthen the Th1 helper reaction and reduce the Th2 influence.The strains K2-Lb6 (deposition number DSM 22829), K6-Lb4, K7-Lb1(deposition number DSM 22831) and K8-Lb1 (deposition number DSM 22832),on the other hand, strengthen the Th2 response and reduce the Th1influence.

The intensity is shown in three stages. The highest of the observedvalues in each case is designated +++. Correspondingly, ++ indicatesapproximately [⅔] and + corresponds to about [⅓].

In FIG. 2, some of the found strains are entered in a phylogenetic tree,in which some other, adequately well know bacterial strains have beenentered. This phylogenetic tree is based on partial sequences of the 16SrDNA of selected examples of ARDRA-PCR grouping of isolates of theLactobacilli from Kimere compared with the BLAST database. Theevolutionary distances were derived using the UIPMGA method, thebootstrap loader program being based on 500 repetitions. To calculatethe evolutionary distances, the method of maximum combined probabilitywas used. This phylogenetic analysis was performed in MEGA4 according toTamura et. al. 2007.

FIG. 3 shows the band patterns of all 10 strains presented here obtainedby the PFGE process-pulse field gel electrophoresis. From FIG. 3, itimmediately becomes clear that the individual band patterns differ fromone another significantly. All the bacteria that are analysed by thePFGE method, and which show precisely this band pattern, are therebyclearly identified as this strain of the respective genus.

The method of typifying microorganisms by PFGE has been known since 1984and is part of the recognized prior art. With the extremely low effortof depositing a single image, it permits the unambiguous identificationof the respective bacterial strain.

FIGS. 4A to 4C show the tolerance of the strains to bile salt.

FIG. 4A shows the number of the surviving strains on exposure to cattlebile with increasing concentration of 0.3%, via 0.5%, 1% and 2%, up to3% cattle bile concentration (w/v).

In FIG. 4B, by measurement of the absorption of the samples at 620 nm,the specific growth of the strains in 0.3% cattle bile solution isplotted against time. A somewhat similar behaviour is shown for allstrains.

FIG. 4C shows the results of an, in principle, identical measurement,but with 3.0% concentration of cattle bile. A particularly rapid growthof the strain K4-Lb6 (deposition number DSM 22830), which is veryobviously highly robust with respect to bile salt, is shown here.

In FIG. 5, the resistance of the strains to very acid milieu is shown,specifically for pH 2 and pH 3. The cells were incubated in 0.35% NaCl(containing 3 g I<−I> pepsin) and adjusted to pH values of 2, 3 and 6.5for 3 h at 37[deg.]C. in each case. The surviving cells were counted onan MRS agar plate at 37[deg.]C. for 48 to 72 h. The values at pH 6.6defined as reference value with 100% and the cells surviving at pH 2 andpH3 were compared with this reference value.

FIGS. 6A to 6C show Th1 and Th2 cytokine production of human blood cells(PBMCs) after co-incubation in vitro with Kimere Lactobacilli strains,specifically with and without stimulation by superantigenicStaphylococcus enterotoxin A (SEA). As an effect of the bacterialstrains on the immune system and on the shift of the Th1/Th2 ratio, FIG.6A shows the production of interferon-[gamma] and FIG. 6B shows theproduction of interleukin-4 and FIG. 6C shows the resulting ratio ofinterferon-[gamma] and interleukin-4.

1-18. (canceled)
 19. Method for examining the identity of the strains,characterized in that the effect-mediating genes have a strain-specificpattern of the suppressive subtractive hybridization (SSH).
 20. Methodaccording to claim 19, characterized in that the part of SSH can bedetermined by pulse field gel electrophoresis. 21-30. (canceled)